Preliminary characterization of EcoRI-DNA co-crystals: incomplete factorial design of oligonucleotide sequences

A full understanding of the sequence specificity of EcoRI endonuclease requires structural information on complexes where the DNA contains one 'incorrect' base pair; historically, these sites are referred to as EcoRI* sites. They are inherently asymmetric, unlike the canonical EcoRI site, GAATTC, which possesses a twofold axis of rotational symmetry. All previously determined DNA-EcoRI complexes incorporated this symmetry axis into the space group, requiring the design of 'new' oligonucleotides to produce an asymmetric unit appropriate to an EcoRI* complex. The incomplete factorial approach of Carter & Carter [Carter & Carter (1979). J. Biol. Chem. 254, 12219-12223.] was used to design the DNA sequence. Factors included the location and type of EcoRI* substitution and the length and AT richness of the sequences on both sides of the RI site. Co-crystals were obtained with several sequences, including one with TCGTGGACTTCGTG. Diffraction data were collected from one crystal of this complex to 3.2 A resolution; the unit-cell parameters are a = b = 123.8 and c = 148.9 A and the space group is P3(2)21. Unit-cell and space-group information was also obtained for the EcoRI* sites AAATTC, GGATTC and GAGTTC. These experiments demonstrated the need for a rapid, economical method that would distinguish DNA-protein co-crystals from crystals of protein only. This can be readily achieved with a single small crystal and a staining method based on methylene blue and methyl violet, which stain DNA and protein, respectively.